Invasive aspergillosis (IA) is a leading cause of morbidity and death in immunocompromised patients, with over 30 million individuals at risk for IA and at least 200,000 IA cases developing worldwide each year. Despite the development of potent triazole antifungal drugs for the treatment of IA, IA-associated mortality is still 22-40%. Timely and accurate diagnosis of IA, in conjunction with prompt initiation of appropriate antifungal therapy, improves patient outcomes, but is challenging due to the shortcomings of existing diagnostics. Due to the limitations of IA diagnostics and the potentially severe clinical consequences of not treating IA promptly, clinicians routinely prescribe empiric antifungal therapy to patients with suspected IA, exposing many patients who ultimately do not have IA to unnecessary antifungal drugs, with their toxicities and costs. A major unmet need in the care of immunocompromised patients is a diagnostic that can identify IA rapidly and accurately, without requiring invasive procedures. To address this unmet need, we propose further development of a novel microfluidic gas chromatography-differential mobility spectrometry (GC-DMS) breath analysis device for the rapid, noninvasive diagnosis of IA, based on the scientific premise that patients with IA have a unique Aspergillus volatile sesquiterpene secondary metabolite signature in their breath. We have demonstrated this premise in an initial proof-of-concept study and subsequent validation study in patients with suspected invasive fungal disease. Combining our expertise in engineering, diagnostic product development, and breath analysis, we have developed an integrated, fully functioning GC-DMS Microanalyzer prototype for rapid bedside breath analysis, successfully detecting these breath sesquiterpene metabolites in patients with IA using this device. The goal of this Fast Track SBIR proposal is further optimization and development of the GC-DMS Microanalyzer breath analysis platform for the rapid diagnosis of IA. The objective of phase I is to optimize the preconcentration of sesquiterpene metabolites in the Microanalyzer device, lowering its limit of detection for these analytes and evaluating its sensitivity in a set of patients with suspected invasive fungal disease. In phase II, aims are to: (1) complete Microanalyzer diagnostic device development for IA under design control, incorporating general and special control measures, (2) develop and validate GC-DMS peak-finding algorithms for the automated identification of the IA breath signature, and (3) perform comprehensive analytical validation studies for this GC-DMS Microanalyzer breath test for IA. Upon successful completion of these objectives, the Microanalyzer breath test for IA will be ready for evaluation in a multicenter clinical validation study for FDA 510(k) clearance and CE mark certification. This diagnostic device will transform the care of immunocompromised patients at risk for IA, reducing diagnostic delays, facilitating early, appropriate antifungal treatment, and improving clinical outcomes in these patients.